Insights over the past decades have demonstrated the importance of mechanical cues in cellular function. However, few tools exist to measure cell-generated forces, and many of these have significant limitations: cells must be plated onto a 2-D surface;cells must be plated onto soft, deformable substrates;and cell forces cannot be measured in multicellular, in vivo environments. The goal of the current proposal is to develop a biosensor protein which would effectively convert a mechanical signal to a fluorescence signal. Changes in the fluorescence signal will be measurable in a wide range of environments, including 3-D assays, substrates of varying stiffness, and in vivo systems. This biosensor protein will be constructed of two domains: a cell-binding domain and a force-transducing domain. The cell- binding domain will consist of a fragment of the extracellular matrix protein, fibronectin, that has been shown to mediate cell adhesion. The force-transducing domain will consist of a length of unstructured polypeptide, flanked on either side by fluorescent proteins. A phenomenon known as Forster Resonance Energy Transfer (FRET) occurs between the two fluorescent proteins when they are in close proximity. This energy transfer diminishes as the two are pulled further apart, and can be measured on a fluorescence microscope. The FRET signal can be used to quantify the end-to-end distance of the polypeptide that links them. The force applied to the unstructured polypeptide can be calculated using mathematical models of polymer dynamics. The relationship between the applied force and the FRET signal will also be measured experimentally using shear flow assays and magnetic field assays. Subsequently, the force biosensor will be used to measure cell-generated forces, with resolution at the level of single integrin bonds. PUBLIC HEALTH RELEVANCE: Cells generate contractile forces that pull on surrounding tissues and cells. There is increasing evidence that the mechanical environment plays an important role in the function and growth of tissues;for example, the tissue of metastatic tumors is significantly stiffer than surrounding healthy tissue;in the liver, stellate cells that drive cirrhosis can only thrive when they have stiff tissue surrounding them;and the fate of mesenchymal stem cells can be directed towards bone, brain, or muscle simply by altering the mechanical properties of the surface to which they are attached. However, limited tools exist to measure these forces. The goal of the current proposal is to develop a protein biosensor that converts mechanical signals into fluorescence signals, allowing for measurement of these cell-derived forces in a wide range of settings.